All lanes : Anti-ATM (phospho S1981) antibody [EP1890Y] (ab81292) at 1/50000 dilution (purified)

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate, untreated
Lane 2 : HEK-293 cell lysate, treated with Doxorubicin

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 351 kDa
Observed band size: 370 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human hepatocellular carcinoma tissue labelling ATM (phospho S1981) with purified ab81292 at 1/70. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, an HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500).

Negative control using PBS instead of primary antibody.

Counterstained with hematoxylin.

Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma) cells labeling ATM (phospho S1981) with purified ab81292 at 1/60 dilution (10 µg/mL) (red).

Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labeling ATM with unpurified ab81292.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human tonsil tissue labeling ATM with unpurified ab81292.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labeling ATM with unpurified ab81292.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human endometrial carcinoma tissue labeling ATM with unpurified ab81292.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gastric carcinoma tissue labeling ATM with unpurified ab81292 at a 1/100 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

ATM was immunoprecipitated from HEK-293 (Human embryonic kidney epithelial cell) treated with Doxorubicin whole cell lysate with ab81292 at 1/30 dilution (5 µg in 1 mg lysates). Western blot was performed from the immunoprecipitate using ab81292 at 1/2000 dilution. An anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG was used as secondary antibody at 1/1500 dilution.
Lane 1: HEK-293 treated with Doxorubicin whole cell lysate 10 µg (Input).
Lane 2: ab81292 IP in HEK-293 treated with Doxorubicin whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab81292 in HEK-293 treated with Doxorubicin whole cell lysate.
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 3 minutes.

Dot blot analysis of ATM peptides using ab81292 at 1/000 dilution followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody (ab97051) at 1/100,000 dilution.

Blocking and diluting buffer was 5% NFDM/TBST, exposure time 3 minutes.

Lane 1: ATM (pS1981) phospho peptide

Lane 2: ATM non-phospho peptide

Lane 3: ATM (pS428) phospho peptide